鲤IGF2b基因5''侧翼区序列克隆及不同鲤该区域GC富集区的甲基化分析

鲤(Cyprinus carpio)胰岛素样生长因子2b(insulin-like growth factor 2b,IGF2b)基因为鲤IGF基因家族重要的成员,已有的研究表明5'侧翼区序列对其功能的发挥有重要作用。因此,采用基因组步移法获取5'侧翼区序列,通过亚硫酸盐修饰的方法分析不同品种该区域GC富集区的甲基化状况。本文成功从鲤基因组DNA中获得695 bp的IGF2b 5'侧翼区序列。已获取的IGF2b 5'侧翼区序列经过TFSEARCH分析后发现了多个转录因子结合位点和TATA框,通过BLAST比对发现,鲤IGF2b 5'侧翼区DNA序列与斑马鱼(Danio rerio)IGF2b基因相比,相似性为87%。检测该区域富含GC的序列17个位点,发现建鲤(C.c.var.jian)有2个个体的3个位点出现甲基化,而黄河鲤(C.c.haematopterus)只有1个个体1个位点出现甲基化,这说明2个品种鲤在这个区域出现甲基化修饰的几率低,也验证了这2品种在该基因该区域GC富集区是稳定的。     

Cloning of IGF2b 5'Flanking Region and Methylation Analysis of Its GC-rich Sequence in Common Carp (Cyprinus carpio)

鲤(Cyprinus carpio)胰岛素样生长因子2b(insulin-like growth factor 2b,IGF2b)基因为鲤IGF基因家族重要的成员,已有的研究表明5’侧翼区序列对其功能的发挥有重要作用.因此,采用基因组步移法获取5′侧翼区序列,通过亚硫酸盐修饰的方法分析不同品种该区域GC富集区的甲基化状况.本文成功从鲤基因组DNA中获得695 bp的IGF2b 5'侧翼区序列.已获取的IGF2b 5'侧翼区序列经过TFSEARCH分析后发现了多个转录因子结合位点和TATA框,通过BLAST比对发现,鲤IGF2b 5'侧翼区DNA序列与斑马鱼( Danio rerio) IGF2b基因相比,相似性为87％.检测该区域富含GC的序列17个位点,发现建鲤(C.c.var.jian)有2个个体的3个位点出现甲基化,而黄河鲤(C.c.haematopterus)只有1个个体1个位点出现甲基化,这说明2个品种鲤在这个区域出现甲基化修饰的几率低,也验证了这2品种在该基因该区域GC富集区是稳定的.     

异源四倍体鲫鲤、三倍体湘云鲫和它们父母本线粒体DNA 12S rRNA基因遗传变异的分析

采用质粒克隆测序方法,获得了异源四倍体鲫鲤5个个体、异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体及红鲫、湘江野鲤和日本白鲫各1个个体的线粒体DNA 12S rRNA基因的全序列。经对比发现,异源四倍体5个个体共享2种单元型,异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体以及红鲫、湘江野鲤和日本白鲫各1个个体分别共享1种单元型。用MEGA 1.0 软件分析了它们的碱基组成和核苷酸序列差异,用邻接法构建系统进化树。它们间的序列同源性在95%~99%之间,异源四倍体鲫鲤、三倍体湘云鲫和它们母本（分别为红鲫和日本白鲫）之间的序列同源性大于异源四倍体鲫鲤、三倍体湘云鲫和它们父本（分别为湘江野鲤和异源四倍体鲫鲤）之间的序列同源性,结果表明：异源四倍体鲫鲤和三倍体湘云鲫在线粒体DNA 12S rRNA基因上具有母性遗传特征。本研究另一值得注意地方的是异源四倍体鲫鲤经过9代(F3-F11)繁殖后,在5个个体中发现了2种单元型,说明在四倍体基因库中存在遗传多样性,为四倍体基因库的繁殖、保护和种群复壮提供了一些有价值的信息。     

鲤春病毒糖蛋白(G)基因的分离及同源性比较

通过RT-PCR和巢氏PCR方法从疑似鲤春病毒(SVCV)侵染的镜鲤肝组织中获得了鲤春病毒糖蛋白(G)基因。通过序列测定与分析,所获得的鲤春病毒糖蛋白(G)基因由606个核苷酸组成,编码一个由202个氨基酸组成的糖蛋白。通过NCBIblast与9个来自不同国家或地区的鲤春病毒糖蛋白(G)基因序列比对分析,发现获得的鲤鱼春季病毒糖蛋白G基因DNA序列与美国分离的AY527273株同源性最高为99.8%,与中国分离的AY842485株同源性次高为98.7%,与英国分离的两个序列SVI538065和SVI538066株的同源性为98.2%,而与其它国家的分离株如SVU18101、SVI318079、SVI538061、SVI538062、SVI538063的同源性最差,仅为89.4%~90.0%。氨基酸同源性分析结果与DNA同源性分析结果一致,所获得的鲤春病毒糖蛋白G基因氨基酸推导序列与其它9个分离株的氨基酸同源性在89.6%~99.5%之间。对SVCV不同分离株遗传变异和进化关系的分析为下一步开展SVCV快速诊断方法的研究和疫苗的研制奠定了基础。     

鲤咽齿个体发生及其与近缘种间关系的比较研究

鲤具鲤科咽齿个体发生的共同性征,又呈现其种的独特性状。主要特性：（１）主列齿相连３齿胚几乎同时出现,同时脱落,形成３个相连齿种同时置换的特殊模式：（２）主列齿发生中曾出现４枚齿,替换３代后,Ａ４齿退化消失,最终齿式１．１．３－３．１．１,成为迄今３列咽齿的鱼类中齿数最少的种类。鲤与其近缘种间系统关系比较结果：依最近缘属顺次排列为中鲤,原鲤,Ｂａ鲤;而最近缘的亚科为Ｂａ亚科。     

与“全红”瓯江彩鲤体色相关的SRAP及SCAR分子标记

利用相关序列扩增多态性(Sequence Related Amplified Polymorphism,SRAP)技术分析"全红"和"粉玉"瓯江彩鲤,筛选与瓯江彩鲤体色相关的分子遗传标记。从88个SRAP引物组合筛选出的12个引物组合共获得扩增条带104个,并筛选出1个SRAP特异扩增带,即"全红"瓯江彩鲤家系SR2,7173 bp带。该条SRAP特异扩增条带经回收、克隆和测序,并将测序结果进行BLAST分析,发现该片段在GenBank中与斑马鱼的POl多蛋白基因和尿红素基因有较高的同源性。根据序列信息分别设计了4对正、反向引物(22—26 bp)。用4对引物分别在"全红"瓯江彩鲤F2和"粉玉"瓯江彩鲤F2群体中进行PCR扩增,仅发现SC-3(154 bp)能够在"全红"瓯江彩鲤群体中特异扩增,而且在"粉玉"瓯江彩鲤F2群体中未出现此扩增带。采用大样本对该SC-3标记进行验证,结果发现,在"全红"瓯江彩鲤群体中呈现阳性,而在"粉玉"瓯江彩鲤群体中为阴性,可以区分这两种群体。因此SC-3标记可以作为"全红"瓯江彩鲤群体一个重要的分子遗传特征指标,为进一步进行分子标记辅助育种奠定了基础。     

鲤和鲫线粒体(mtDNA)全基因组分析

为研究鲤(Cyprinus carpio)和鲫(Carassius auratus) mtDNA上的基因分布特征,丰富鱼类mtDNA数据库。本研究通过PCR扩增和测序,获得了鲤和鲫mtDNA基因组序列,经比对分析表明,鲤和鲫的mtDNA序列全长均为16 596 bp,共有13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个D-Loop区。鲤和鲫的mtDNA序列中(A+T)百分含量分别为57.2%和57.1%,其碱基组成均具有一定的A/T碱基偏向性。N-J系统发育树分析表明,鲤和鲫与琵琶湖鮈亲缘关系最近,与达氏深水尾魟亲缘关系最远。本研究揭示了鲤和鲫mtDNA遗传变异特征及基因分布规律,为鱼类遗传资源保护及开发利用提供了参考依据。     

从江及周边地区稻田鲤的遗传结构及系统发育关系

为探究贵州从江及邻近的榕江、黎平和锦屏以及广西三江、湖南的通道和靖州等地稻田鲤Cyprinus carpio Linnaeus的遗传结构及系统关系, 研究基于mtDNA Cyt b基因测序技术对6个地理群体进行了分析。447尾稻田鲤中共界定出29种Cyt b基因序列单倍型, 结合NCBI中鲤同源序列的系统发育和序列比对分析表明, 从江及邻近苗侗族地区稻田鲤是一个含欧亚2大谱系、3个鲤亚种和多个母系血统成分的混合群体, 主体类群是华南鲤C. carpio rubrofuscus, 同时混有欧洲鲤C. carpio carpio和远东鲤C. carpio haematopterus, 遭受国内外鲤养殖品种的遗传渗析。遗传多样性分析显示, 欧洲鲤亚种群体为低H_d低π, 遗传多样性贫乏, 远东鲤和华南鲤2个亚种群体呈高H_d低π特点。华南鲤6个稻田群体除锦屏为低H_d低π外, 其余都为高H_d低π, 从江的H_d最高, 黎平的π最高。遗传分化和分子方差(AMOVA)分析显示, 谱系间和亚种间存在显著的遗传分化(P<0.001), 锦屏和三江与其他群体存在中等-高度的分化等级, 其余群体间分化较弱, 群体间变异贡献率为16.69%, 群体内为81.14%。综合分析表明, 从江及邻近地区稻田鲤群体间存在有限的遗传交流, 群体内和群体间有不同的遗传结构。研究丰富了该地区稻田鲤遗传背景资料, 可为湘黔桂交界苗侗族地区稻田鲤的种质资源鉴定与保护提供理论依据。     

人工复合三倍体锂与亲本相对DNA含量及倍性分析

本文通过显微荧光光度术了人工复合三倍体锂的红细胞及亲本红鲤,红鲫与散鳞镜鲤个体的红细胞和精子的相对ＤＮＡ含量。结果表明;每一种亲本的精子ＤＮＡ含量是其红细胞ＤＮＡ含量的二分之一,人工复合三倍体鲤ＤＮＡ含量等于三个亲本精子ＤＮＡ含量之和,为三个亲本血液红细胞ＤＮＡ含量之和和二分之一。     

鲤在低温胁迫下肝胰腺转录组测序分析

检测低温耐受与低温敏感鲤品种低温胁迫下的差异表达基因,探究鲤低温适应的分子应答过程。低温胁迫后对松浦镜鲤及荷包红鲤肝胰腺进行转录组测序,并对表达差异基因进行功能、通路富集分析。通过差异表达分析,共获得10 521个差异表达基因,其中5 246个基因仅在低温耐受品种发生表达变化。功能、通路富集分析将差异基因显著富集在包括糖酵解/糖质新生代谢途径在内的144个代谢途径。选取5个基因进行qRT-PCR验证,定量结果与RNA-seq结果相关性达0.89。研究结果表明低温胁迫会显著改变鲤肝胰腺的转录活动,编码糖酵解/糖质新生途径的多个关键酶基因在低温耐受品种的表达量显著高于低温敏感品种,推测这些基因功能有助于其长期低温适应。     

Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA

The complete cytochrome b and the control region of mtDNA (about 2070 bp in total) of 10 strains belonging to three subspecies of the common carp, including three wild subspecies (the Yangtze River wild common carp – Cyprinus carpio haematopterus , Yuanjiang River wild common carp – Cyprinus carpio rubrofuscus and Volga River wild common carp – Cyprinus carpio carpio ) and seven domestic strains (Xingguo red carp, Russian scattered scaled mirror carp, Qingtian carp, Japanese Koi carp, purse red carp, Big-belly carp, German mirror carp) were sequenced. Phylogenetic analysis indicated that the 10 strains form three distinct clades, corresponding to C. c. haematopterus , C. c. rubrofuscus and C. c. carpio respectively. Purse red carp, an endemic domestic strain in Jiangxi province of China, showed a higher evolution rate in comparison with the other strains of C. c. haematopterus , most probably because of intensive selection and a long history of domestication. Base variation ratios among the three subspecies varied from 0.78% (between C. c. haematopterus and C. c. rubrofuscus ) to 1.47%(between C. c. carpio and C. c. rubrofuscus ). The topography of the phylogenetic tree and the geographic distribution of three subspecies closely resemble each other. The divergence time between C. c. carpio and the other two subspecies was estimated to be about 0.9 Myr and about 0.5 Myr between C. c. haematopterus and C. c. rubrofuscus . Based on phylogenetic analysis, C. c. rubrofuscus might have diverged from C. c. haematopterus .     

鲤鱼不同品种（系）红血球抗原特异性的初步研究

用血球凝集和血清吸收试验的方法,对若干个鲤鱼品种及鲤鱼人工雌核发育系的红血球中的某些同种异型抗原纯合性进行了初步研究。①以抗雌核发育红鲤抗血清(abs／R)为试剂,三个雌核发育系(R8305,R8413,RMGF1)的RBC反应值分别为1521—2360,1521-2028,853—1521,普通红鲤、镜鲤、白鲢的反应值在80—782。②abs／8305,与abs／8413经靶细胞相互吸收后的残留活性显著降低(均为320),而以白鲢RBC吸收后抗血清活性不变。作者认为这部分抗原在鲤鱼种以下的品种和品系中具有特异性,可考虑将其作为鱼类品系鉴定的一种手段。     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Genetic variation analysis within and among six varieties of common carp (Cyprinus carpio L.) in China using microsatellite markers

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (p < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F(st) distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.     

Genetic Variation Analysis within and among Six Varieties of Common Carp (Cyprinus carpio L.) in China Using Microsatellite Markers

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles ranging from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (P < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F _st distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.     

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp ( Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp ( Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.     

Comparative analysis of intermuscular bones in three strains of common carp

The objective was to document the variations in the intermuscular bone counts among three carp strains: mirror carp, hybrid carp [Boshi carp (Cyprinus pellegrini) × Heilongjiang carp (Cyprinus carpio)], and a cold‐resistant strain of Hebao carp. The intermuscular bone counts, lengths, and weights were obtained from 146 fish; the bone count ranged from 55 to 110 (mean 92.85) among the three strains. Mirror carp had the lowest number of intermuscular bones and a higher coefficient of variation in counts relative to the other two species. There was no relationship between the intermuscular bone counts to standard length or body length. Similarly, there was no difference in the intermuscular bone count or shape between the left and right sides of the body. However, the count was significantly higher (P < 0.01) in the anterior region (snout to cloaca: 54% of the total count) than in the posterior region (cloaca to base of caudal fin: 46% of the total count). There was a significant difference between strains (P < 0.0001), but not between age classes; a significant difference was also observed in the posterior region counts among strains, but not in the anterior region (P < 0.0001). Similarly, the difference in counts in the posterior region was primarily related to differences in the counts of three of the six types: non‐forked (I), tree‐branch and two‐end‐bi‐fork type. Our results suggest that selective breeding protocols should target a reduction in the number of intermuscular bones in the posterior region, particularly in mirror carp.     

Haematological profile of common carp spawners of various breeds

The goal of this study was to determine the basic haematological profile of common carp (Cyprinus carpio L.) spawners of eight breeds reared under identical conditions and sampled in spring after overwintering. Significant differences were found among the breeds for haemoglobin level (Hb), haematocrit value (PCV), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values. The number of red blood cells (RBC), mean corpuscular haemoglobin concentration (MCHC), number of leukocytes (WBC) and leukocyte differential count (WBC DIFF) did not differ significantly among the respective breeds. The highest Hb, PCV and MCH values were found for Ropsha scaly carp (ROP) and Amur wild carp (AS) (Hb 109 ± 17 and 106 ± 15 g l^?1; PCV 0.40 ± 0.06 and 0.40 ± 0.06 l l^?1; MCH 69 ± 7 and 69 ± 9 pg, respectively). The AS specimens and breeds that originated [ROP, Ukraine scaly carp (US), and Northern mirror carp (M72)] showed significantly higher values of Hb (P < 0.01), PCV (P < 0.01), MCV (P < 0.05) and MCH (P < 0.05), compared to the other four breeds: Hungarian mirror carp (M2), Israeli mirror carp (Dor 70), South Bohemian mirror carp (BV) and Tata scaly carp (TAT). Males showed significantly higher PCV, Hb and RBC values within individual breeds. This study demonstrated that Amur wild carp and breeds originating from it had significantly higher values of erythrocyte profile in comparison with the other breeds studied.     

银鲫复合种外源遗传物质整入的RAPD分析

在优化ＲＡＤＰＤ检测条件的基础上,采用４０对随机引物,比较分析了银复合物,异育银鲫和兴国红鲤相互间扩增ＤＮＡ片段的异同性。总的来说,银鲫复合种和异育银鲫具有基本一致的扩增产物,而与兴国红鲤的扩增产物多数不同,相似率分析表明,银鲫复合种与兴国红鲤之间的相似率为３１．６％,异育银鲫与兴国红鲤之间的相似率为２８．６％。在分析中,除发现银鲫复合种,异育银鲫与兴国红鲤间共有扩增片段外,还发现了银鲫复合种与兴